Mice

NOD/ShiLtJ (#001976) and NOD Scid Gamma (NSG; #005557) mice were purchased from Jackson Laboratories. B6.Aire^−/− and NOD.Aire^−/− were kindly provided by Diane Mathis and Christophe Benoist (Harvard University, Boston, MA). Adig (Gardner et al., 2008) mice were kindly provided by Mark S. Anderson (University of California, San Francisco, CA). B6.Aire^Y86C, B6.Aire^C313X, B6.Aire^C442G, NOD.Aire^V303M, and NOD.Aire^C313Y mice were generated at the transgenic facility at the Weizmann Institute of Science, using CRISPR/Cas9 genome editing in isolated one-cell embryos from C57Bl/6 or NOD mice, respectively. Generating these mice on a NOD background was attempted for most strains, but was not always successful even after several attempts as the number of oocytes successfully harvested and fertilized following in vitro fertilization was much lower compared with C57Bl/6. Therefore, the C57Bl/6 background was used to generate some of the mutations. Genotyping of mice with point mutations was conducted using custom TaqMan SNP Genotyping assays (Table S4). The B6.Aire^C442G mice were genotyped by Sanger sequencing of the relevant locus as an adequate TaqMan SNP Genotyping assay could not be generated.

As NOD mice have smaller thymi yielding fewer TECs, NOD.Aire^+/C313Y males were bred with WT C57Bl/6 females (Envigo) for ChIPseq experiments. F1 progeny were used for sorting mTEChi to obtain more cells per mouse.

The early onset of autoimmunity in NOD.Aire^+/C313Y mice hampered their ability to breed with other NOD.Aire^+/C313Y mice. Therefore, WT NOD/ShiLtJ mice or NOD.Aire^+/− mice were used to produce both NOD.Aire^+/C313Y and NOD.Aire^C313Y/– mice. All mice were maintained and bred under specific pathogen–free conditions at the Weizmann Institute of Science’s animal facility and were provided with standard rodent chow and autoclaved water ad libitum. 4–7-wk-old mice were used in all experiments unless stated otherwise, and all handling and experimentation were conducted in accordance to the guidelines of the Weizmann Institute of Science Institutional Animal Care and Use Committee (#10060119-2). All control mice used were littermates of the relevant mutant strain.